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EP2552467A1 - Procédé d'extraction d'isothiocyanates - Google Patents

Procédé d'extraction d'isothiocyanates

Info

Publication number
EP2552467A1
EP2552467A1 EP11712387A EP11712387A EP2552467A1 EP 2552467 A1 EP2552467 A1 EP 2552467A1 EP 11712387 A EP11712387 A EP 11712387A EP 11712387 A EP11712387 A EP 11712387A EP 2552467 A1 EP2552467 A1 EP 2552467A1
Authority
EP
European Patent Office
Prior art keywords
mustard
essential oil
solvent
isothiocyanate
oil
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11712387A
Other languages
German (de)
English (en)
Inventor
Athula Ekanayake
Scott Alan Vandiest
Jeffrey John Kester
Paul Henry Zoutendam
Jairus R. D. David
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Procter and Gamble Co
Original Assignee
Procter and Gamble Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Procter and Gamble Co filed Critical Procter and Gamble Co
Publication of EP2552467A1 publication Critical patent/EP2552467A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01147—Thioglucosidase (3.2.1.147), i.e. myrosinase

Definitions

  • Embodiments of the present invention relate to processes of extracting isothiocyanates from plant materials. More particularly, but not exclusively, embodiments of the present invention relate to processes of extracting essential moisture sensitive isothiocyanates from mustard seeds and essential oils of mustard seeds.
  • Consumer products can provide a hospitable environment for rapid microbial growth. Such exposure can, and frequently does, result from inadvertent microbial inoculation of the product during manufacturing or packaging. Spoilage microorganisms, for example in food products, can then rapidly proliferate by feeding on nutrients provided by the product.
  • Preservatives such as sorbates, benzoates, organic acids, and combinations thereof have been used in various products, particularly foods and beverages, to provide some degree of microbial inhibition. However at levels effective to inhibit microbial growth, some of these preservatives can contribute off-flavors in the product, thus making the product undesirable for its intended purpose. Similarly, natural preservatives, such as natamycin, are frequently used in food and beverage products to inhibit microbial growth. Unfortunately, while these natural preservatives may be effective against either yeast or bacteria, they may not be effective against both.
  • the essential oil of mustard plants which contain isothiocyanates, exhibits an antibacterial and antimycotic effect in oral therapies and on certain foods. See e.g., Sekiyama et al., U.S. Patent No. 5,334,373, assigned to Nippon Sanso Corp., issued August 2, 1994; and Madaus et al., U.S. Patent No. 3,998,964, issued December 21, 1976.
  • the isothiocyanate compounds in mustard essential oils are the active agents that provide the antimicrobial effect.
  • the essential oil derived from white or yellow mustard plants (Sinapis alba or Brassica alba), also provides the foregoing antibacterial and antimycotic benefits.
  • isothiocyanate compounds are effective antimicrobial agents at relatively low usage levels.
  • the principal isothiocyanate present in the white mustard essential oil, 4-hydroxybenzyl isothiocyanate (4-HBITC) is a moisture sensitive compound that begins to degrade (i.e. hydrolyze) within hours of being exposed to moisture. When degraded, the 4-hydroxybenzyl isothiocyanate forms, among other compounds, 4-hydroxybenzyl alcohol.
  • white mustard essential oil is not volatile at atmospheric pressure and requires that it be extracted out of the seeds by the use of a solvent or a method such as supercritical fluid extraction.
  • white mustard essential oil unlike most of the other plant essential oils, is relatively unstable, especially so when exposed to moisture. This instability imposes the additional condition that when the essential oil is generated that it be extracted from the mustard seed and soon stabilized thereafter to maintain its antimicrobial properties.
  • the mustard processing industry makes use of the white mustard flour primarily while the essential oil is largely ignored.
  • the ground mustard flour is subjected to a thermal deactivation step.
  • the enzyme myrosinase which catalyzes the formation of 4-hydroxybenzyl isothiocyanate from its precursor 4-hydroxybenzylglucosinolate, also known as sinalbin, is intentionally deactivated so that the essential oil does not form when the flour is mixed with moist food products such as meat and sausage.
  • 4-hydroxybenzyl isothiocyanate is not currently available commercially, whether as a natural product or as a pure chemical.
  • white mustard essential oil has not been widely known or widely utilized in the art for its antibacterial and antimycotic effect.
  • the present inventors have surprisingly discovered that, in one embodiment, by generating the white mustard essential oil by adding water to defatted ground mustard seed, extracting the white mustard essential oil using solvents or supercritical fluids, drying the essential oil by removing the solvent and residual moisture, and then intimately blending the resulting white mustard essential oil with a hygroscopic carrier, the moisture sensitive isothiocyanate compounds contained therein can be stabilized.
  • the blend of white mustard essential oil with a hygroscopic carrier is, thereafter, capable of being used as an effective antibacterial and antimycotic agent for solid food products.
  • One embodiments relates to a process for producing an essential oil. It can include providing a plant material comprising a glucosinolate precursor and myrosinase enzyme, wherein the plant material comprises a fixed oil; reducing the particle size of the plant material to produce a powder; activating the myrosinase enzyme by using a water solvent and a promoter to form a slurry, wherein the myrosinase enzyme catalyzes the production of an essential oil comprising an isothiocyanate from the glucosinolate precursor; and separating the slurry into an essential oil and a residual solvent.
  • Another embodiment relates to a process for producing an essential oil. It can include providing mustard seed comprising a precursor sinalbin and myrosinase enzyme, wherein the mustard seed comprises a fixed oil; optionally, reducing the fixed oil content of the mustard seed; reducing the particle size of the seed to produce a mustard powder; activating the myrosinase enzyme by using a water solvent and a promoter to form a slurry, wherein the myrosinase enzyme catalyzes the production of an essential oil comprising an isothiocyanate from the sinalbin precursor; and separating the slurry into an essential oil and a residual solvent.
  • the essential oil can be a white mustard essential oil, which can include a moisture sensitive isothiocyanate compound, which can be 4-hydroxybenzyl isothiocyanate.
  • compositions and processes herein may comprise, consist essentially of, or consist of any of the features or embodiments as described herein.
  • the term "antimicrobial effect” means that the product inhibits growth of, eliminates, and/or otherwise decreases the presence of microorganisms such as, for example, yeast, bacteria, mold, and/or fungus, preferably yeast and/or bacteria.
  • essential oil refers to the set of all the compounds that can be distilled or extracted from the plant from which the oil is derived and that contributes to the characteristic aroma of that plant. See e.g., H. McGee, On Food and Cooking, Charles Scribner's Sons, p. 154 - 157 (1984).
  • the essential oil preferably originates from the white or yellow mustard plant (Sinapis alba or Brassica alba), which is capable of producing a moisture sensitive isothiocyanate compound, and more specifically, 4-hydroxybenzyl isothiocyanate (4-HBITC).
  • moisture sensitive means that the isothiocyanate compound degrades in the presence of water. This degradation proceeds via a hydrolysis reaction, thereby leading to a reduction in level of the active isothiocyanate antimicrobial agent with time of storage in the presence of water.
  • the method for determining moisture sensitivity is set forth in the Test Method section below.
  • Moisture sensitive isothiocyanates are characterized by a reduction in concentration of the isothiocyanate compound of at least about 20% of the starting concentration, when suspended in an aqueous phosphate buffer having a pH of about 3.6, and a temperature of from about 20 °C to about 23 °C, over a 24 hour period.
  • a moisture sensitive compound is 4-hydroxybenzyl isothiocyanate.
  • the term "natural component,” with reference to the corresponding essential oil, refers to a component utilized in the present invention that is obtained from the naturally occurring essential oil.
  • the term "substantially free of means comprise less than about 0.05% by weight (i.e. less than about 500 parts per million).
  • Embodiments of the present invention relate to processes of extracting isothiocyanates from plant materials.
  • embodiments of the present invention relate to processes of extracting oils from plants.
  • Embodiments of the present invention relate to processes of extracting essential moisture sensitive isothiocyanates from mustard seeds and essential oils of mustard seeds.
  • the process comprises extracting essential oils from plants.
  • oils can include white mustard essential oil (WMEO).
  • white mustard essential oils can include isothiocyanate compounds, which can be a moisture sensitive compound.
  • Such compounds have been previously identified as having beneficial antimicrobial activity in food products. See United States Patent No. 7,658,961, assigned to The Procter & Gamble Company.
  • these compounds are often used in combination with the known preservatives benzoic acid, sorbic acid, or salts thereof and/or the moisture sensitive isothiocyanate compound, such as the 4-hydroxybenzyl isothiocyanate in white mustard essential oil, can be combined with a hygroscopic carrier that attracts, absorbs, and binds moisture, without the use of these known preservatives.
  • the moisture sensitive isothiocyanate compound such as the 4-hydroxybenzyl isothiocyanate in white mustard essential oil
  • the constituent comprising the moisture sensitive isothiocyanate compound can be an essential oil, natural component of an essential oil, or synthetic component of an essential oil (all as described in more detail hereinafter) of the white or yellow mustard family (Sinapis alba or Brassica alba).
  • the Brassica family of plants is a small family having about 2000 species and over 300 genera (see e.g. Natural Food Antimicrobial Systems, edited by A.S. Naidu, CRC Press LLC, pp. 399-416, 2000).
  • the constituent comprising the moisture sensitive isothiocyanate compound may be an essential oil, natural component of an essential oil, or synthetic component of an essential oil of any other family of plants which may produce a moisture sensitive isothiocyanate compound.
  • Food Chemistry Edited by O.R. Fennema, Marcel Dekker, Inc., pp. 602 - 603 (1985) and Naturally Occurring Antimicrobials in Food, Council for Agricultural Science and Technology, pp. 31 - 32 (1998).
  • the seeds and/or flowers of any of, for example, a Brassica species may be, homogenized, ground, crushed, pressed, or otherwise damaged to activate one or more precursors (e.g., glucosinolates) of the corresponding essential oil.
  • Isothiocyanate compound production from the oil is known to occur by enzyme catalysis upon, for example, homogenizing, grinding, crushing, pressing, or otherwise damaging the plant, seed, and/or flower thereof. See e.g., Concannon, WO 94/01121, published January 20, 1994 and Brown et al., "Glucosinolate-Containing Plant Tissues as Bioherbicides", Journal of Agricultural Food Chemistry, Vol. 43, pp.
  • the enzyme commonly known to participate in the production of the isothiocyanate compound upon interaction with a glucosinolate is myrosinase, which is also known as thioglucoside glucohydrolase (and having enzyme classification number EC 3.2.3.1).
  • Myrosinase is known to be non-specific for various glucosinolates.
  • the embodiments herein can be related to any plant material that comprises a glucosinolate precursor and myrosinase enzyme, one non-limiting example which is mustard seed.
  • plant materials containing a moisture sensitive isothiocyanate compound is envisioned.
  • the plant materials can include the family Brassicaceae (previously Cruciferae) and can include mustards (Brassica nigra, Brassica juncea, Brassica hirta or Sinapis alba), cabbage (Brassica oleracea), cauliflower (B. oleracea var. botrytis), brussel sprouts (B. oleracea var. gemmifera), broccoli (B. oleracea var.
  • kohlrabi B. oleracea var. gongylodes
  • wasabi or Japanese horseradish
  • canola B. napus
  • turnips B. rapa
  • Additional non-limiting examples include other plant materials such as capers (Capparaceae) and Moringaceae.
  • white mustard essential oil comprising from about 5% to about 35% 4-hydroxybenzyl isothiocyanate (4-HBITC) is provided from white or yellow mustard, Sinapis alba.
  • WMEO comprising from about 10% to about 30% 4-hydroxybenzyl isothiocyanate (4-HBITC) can be produced.
  • WMEO comprising from about 15% to about 27% 4-hydroxybenzyl isothiocyanate (4-HBITC) can be produced.
  • WMEO comprising from about 22% to about 28% 4-hydroxybenzyl isothiocyanate (4-HBITC) can be produced.
  • compositions comprising, however made, white mustard essential oil comprising about 30% to about 35% 4-hydroxybenzyl isothiocyanate (4-HBITC).
  • This active compound in white mustard essential oil, 4-HBITC is a moisture sensitive compound that begins to degrade (i.e. hydrolyze) within hours of being exposed to moisture.
  • 4-HBITC is very susceptible to hydrolysis at neutral pH.
  • white mustard seeds contain its precursor sinalbin, and the enzyme myrosinase (thioglucosidase glucohydrolase).
  • myrosinase When activated by the addition of water, producing a moistened mustard seed, myrosinase catalyzes the degradation of sinalbin resulting in 4-hydroxybenzyl isothiocyanate.
  • the 4-HBITC and other lipid soluble material transfer into the solvent layer and can then be separated from the moistened mustard seed.
  • the solvent can then removed at low temperature under reduced pressure to yield WMEO comprising 4-HBITC.
  • 4-HBITC preparation which can be dry, can then be used as the preservative in accordance with US 6,361,812B1 ; US 6,558,723B2; US 7,105, 190B2 and US 7,658,961, all assigned to The Procter & Gamble Company.
  • the extraction process can comprise the following general steps, all of which are not necessarily required, and all of which are described hereinafter: 1) cold pressing or solvent extracting mustard seeds to remove fixed oil; 2) crushing the reduced fat or fat free mustard seeds to give a defatted white mustard powder; 3) adding the defatted white mustard powder into the solvent and water mixture containing the activator ascorbic acid; 4) letting the myrosinase catalyzed sinalbin hydrolysis reaction happen over a period of time, and allowing the
  • white mustard seeds can be first cold pressed to remove as much as possible of the seed oil without increasing the temperature of the press cake.
  • the press cake temperature can be less than about 70°C to ensure that myrosinase enzyme activity is retained in the press cake.
  • it can be moistened to get myrosinase to be active in catalyzing the hydrolysis of sinalbin to 4-HBITC.
  • ascorbic acid at about 1 millimole can be used an effective activator of the myrosinase enzyme.
  • a solvent can be mixed in with the reacting moist mustard system, which can ensure that the 4-HBITC produced by the reaction will transfer to the lipophilic ethyl acetate layer when utilizing an ethyl acetate solvent.
  • the ethyl acetate layer can be separated.
  • the press cake:water:ethyl acetate ratio can be about 1:0.3:2 to avoid the formation of a stable emulsion and to have sufficient water to allow the enzyme to be active.
  • the ethyl acetate containing the 4-HBITC and the residual mustard oil can be removed from the reaction system by centrifugation and immediately evaporated under vacuum to yield WMEO.
  • drying the ethyl acetate can be avoided.
  • the WMEO can then be kept frozen to avoid the degradation of 4-HBITC.
  • plating the WMEO on maltodextrin as taught in US 7,658,961 can be performed to allow it to be stored at refrigerated or room temperature.
  • the fixed oil content of the mustard seed which can comprise a precursor sinalbin and myrosinase enzyme, can be reduced.
  • the fixed oil content of the mustard seed is about 26% to about 28% by weight.
  • the fixed oil content is comprised of the triglycerides found in the mustard seed.
  • the fixed oil content of the mustard seed can comprise from about 2% to about 10%, or from about 4% to about 9%, or from about 6% to about 8%.
  • Reducing the oil content can comprise mechanical expression, such as cold pressing or solvent extracting mustard seeds to remove fixed oil followed by crushing the reduced fat or fat free mustard seeds to give a defatted white mustard powder.
  • mechanical expression such as cold pressing
  • Cold pressing can generally involve subjecting the thing to be cold pressed to temperature and pressure, as is well known in the art.
  • cold pressing can include subjecting the mustard seed to pressure and temperature as described above and extracting essential oil from the seed without the use of solvent extraction.
  • This embodiment can be termed "oil expressing" and can be without the use of a solvent extraction.
  • the temperature can be kept at or below about 70°C, or below about 50°C. Such temperature can leave the myrosinase active.
  • the oil and pressed cake that is left after extraction of the oil can be processed as disclosed hereinafter.
  • the pressed cake can be crushed, milled, or otherwise processed by reducing the particle size into a mustard powder.
  • the mustard seed can be mechanically expressed, such as by cold pressing.
  • This expressing can produce a pressed mustard seed cake, which includes the essential oil.
  • mustard seed has an oil content of from about 26% to about 28% and a moisture content of form about 6% to about 7%.
  • the myrosinase catalyzed hydrolysis reaction of glucosinolate cannot occur because the moisture content is not sufficient to support enzymatic action.
  • mustard seeds provide a good source for pressing out or expelling the fixed oil.
  • Oil expressing can be done in many different types of machines to produce a defatted powder.
  • a single press screw can press mustard seeds within a press cylinder.
  • the single press screw can include a feeding hopper at one end of the screw containing cylinder, a press head at the other end where the seed is pressed to squeeze out the oil, and a perforated section between the hopper and press head.
  • the perforated section can allow the expressed oil to flow out.
  • Pressed mustard seeds result in a defatted press cake that can be extruded or forced through a nozzle in the press head and can form a continuous cylindrical pellet as it exits the machine.
  • the large thermal capacity of these machines can prevent the temperature of the press cake from rising much above about 60°C to about 65°C, which can be beneficial for pressing mustard seed.
  • the defatted pressed cake can have about 6-8% residual fat and can also contain the precursor sinalbin and the enzyme myrosinase.
  • the temperature that the press cake had experienced during the pressing step is insufficient to denature the myrosinase enzyme.
  • the defatted mustard can then exit the press as a cylindrical pellet that can be milled to a fine powder, which can in one embodiment become the raw material for the next step in the process.
  • a type of screw press called a cage type of press
  • the press cylinder containing the press screw can be slotted from a point close to the feeding hopper to the end of the press cylinder.
  • the press screw can push the mustard seed against these slots to press out the oil, which can exit the press cylinder via the longitudinal slots with some plant debris.
  • the slot spacing and the press screw pressure pieces can be adjusted to give the clearest oil and the defatted press cake with the lowest possible oil content.
  • the long press area generates much friction and consequent heat.
  • the thermal capacity of these types of presses is low and as a result the press cake tends to warm to temperatures as much as about 80°C to about 85°C.
  • the myrosinase activity of the defatted press cake may be adversely affected by this rise in temperature. If the press cake temperature is from about 70°C to about 75°C when it exits from the press, it can be cooled soon after thereby maintaining sufficient enzyme activity to catalyze the hydrolysis of sinalbin when the press cake is wetted.
  • the defatted press cake may contain up to 17% residual fat.
  • Another variation in the use of the cage type of screw press is to divide the mustard seed into two lots.
  • the first of these lots can be pressed so that the press cake temperature does not rise above from about 65°C to about 70°C while the second lot can be pressed to maximize oil removal, irrespective of the press cake temperature.
  • the final residual oil content can be much lower than 17% and, on wetting, the combined press cake and the myrosinase enzyme in the first lot will be active and able to catalyze the hydrolysis of sinalbin from both lots to 4-hydroxybenzyl isothiocyanate.
  • the proportions of the two lots can be changed.
  • mustard seed can be divided into three lots. Two of these lots can be subjected to the higher temperature pressing step while the third lot can be subjected to the lower temperature pressing step thus preserving the enzyme activity of this third lot.
  • the three lots can be re-combined to give a final residual oil content of much less than 17% and an enzyme activity sufficient to catalyze the hydrolysis of sinalbin of all of the lots to 4-hydroxybenzyl isothiocyanate.
  • the powder or pressed cake can then be treated to extract the residual fixed oil and activate the myrosinase enzyme.
  • Such treatment can be performed by adding a solvent and a promoter.
  • Solvents can include water solvents, ethyl acetate, water, and combinations and mixtures thereof.
  • the solvents can be added with the powder or pressed cake in a reaction vessel to form a reaction mixture.
  • the myrosinase enzyme will be activated, and the mustard cake or powder and solvent and promoter form a slurry.
  • the myrosinase enzyme can catalyze the production of an essential oil comprising an isothiocyanate from the sinalbin precursor.
  • the essential oil can be WMEO
  • the isothiocyanate can be a moisture sensitive isothiocyanate, such as 4-HBITC.
  • Solvents that can be used to extract the residual fixed oil of the partially defatted mustard press cake along with the generated 4-hydroxybenzyl isothiocyanate including, but are not limited to, ethyl acetate, hexane, heptanes, methyl pentane, dichloromethane, and chloroform. In one embodiment, ethyl acetate is used. Ethyl acetate is not water soluble and thus can be removed easily from the reaction mixture by a simple centrifugation step.
  • ethyl acetate forms a lower boiling azeotrope with water having the composition 91.53% ethyl acetate and 8.47% water with a boiling point of 70.4°C compared to 77.2°C for pure ethyl acetate.
  • This formation allows the separated ethyl acetate to be removed from the final solvent removal step without a pre-drying step because the added water is also removed by the azeotrope that it forms with ethyl acetate. Drying agents used to remove water from solvents have to be regenerated before reuse and that requires them to be heated and kept under dry conditions before use.
  • ethyl acetate as solvent is the azeotrope that ethyl acetate forms with water reduces the boiling point of ethyl acetate by about 7°C thus allowing a lower temperature of evaporation.
  • the solvent can be mixed with the required amount of water in a reaction tank and the powdered, partially defatted mustard press cake can be added into the tank with mixing to ensure good wetting.
  • the powdered, partially defatted mustard press cake can be first mixed with the dry activator/promoter to ensure homogeneity and wetted with the requisite amount of water in mixing vessel.
  • the solvent can then be added to this mixing vessel and mixed at high speed first to ensure homogeneity and then mixed at lower speed to maintain the slurry in suspension for the reaction to take place and the 4-hydroxybenzyl isothiocyanate to transfer to the solvent.
  • a combination of ethyl acetate and water can be used.
  • the particular amounts of the ethyl acetate and water can be varied.
  • the fat of the defatted mustard press cake content can be relevant to the amount of solvent used.
  • the defatted mustard pressed cake can have as high as 17% fat content.
  • the added water can be maintained at less than about 40% with a maximum ethyl acetate content of about 2 times the weight of the partially defatted mustard press cake used to form a mustard slurry. Such an embodiment can avoid the formation of a stable emulsion.
  • emulsion formation can be avoided in order to be able to separate the ethyl acetate from the reaction mixture by a simple centrifugation step described hereinafter.
  • the proper amount of moisture controls such that the reaction begins and partition between the ethyl acetate and the white mustard powder but not too much to form an emulsion.
  • the defatted white mustard:water:ethyl acetate ratio is selected to provide sufficient water for the myrosinase catalyzed hydrolysis reaction of sinalbin to 4-hydroxybenzyl isothiocyanate to occur efficiently and also to avoid forming a stable emulsion described above when the moistened defatted mustard press cake is mixed with ethyl acetate.
  • the ratio of PDMS:water:ethyl acetate can be about 1:0.4: 1.8.
  • the ratio of defatted mustard press cake to water to ethyl acetate can be 1 part defatted mustard press cake: 0.4 part water: 1.8 parts ethyl acetate, on a weight basis.
  • the ratio of PDMS:water:ethyl acetate can be about 1:0.25:1.5 to about 1:0.5:3, by weight.
  • a promoter can also be used.
  • a promoter is a substance that speeds up the reaction catalyzed by the myrosinase enzyme.
  • the promoter can be ascorbic acid.
  • metal salts could be used.
  • the promoters can be added at between about 0.75 millimole and about 3 millimoles, or about 1.0 millimole and about 2.5 millimoles, 1.5 millimole and about 2.0 millimoles, or about 1.0 millimole.
  • the amount of promoter used allows for the proper activation of the myrosinase enzyme. Lower amounts may not provide for the proper activation of myrosinase enzyme while higher amounts may react with 4-HBITC to give compounds called ascorbinogens, which may reducing the yield of 4-HBITC.
  • Myrosinase or thioglucosidase glucohydrolase (E.C. 3.2.1.147) is an enzyme with a site or sites that can host the promoters, such as the ascorbic acid molecule.
  • the maximum concentration of 4-HBITC in the solvent occurs after about 24 hours in the reaction slurry comprising partially defatted white mustard, water and solvent at room temperature of about 21 °C.
  • the maximum concentration in a similar reaction system is reached within about 3-4 hours and that concentration is maintained for the next 20-22 hours providing a wide enough window to centrifuge and separate the water immiscible organic solvent in large scale production systems.
  • a promoter such as ascorbic acid
  • the partially defatted mustard seed can be directly added to the solvent mixture to form a slurry.
  • the solvent such as an ethyl acetate-water mixture
  • the process can be sped up by avoiding a cumbersome pre-wetting step.
  • PDMS when wetted with water tends to form lumps in contrast to adding to an ethyl acetate-water mixture when the PDMS wets easily and disperses easily to form a slurry that is stirred easily.
  • the slurry can be allowed to react for a specified reaction time.
  • the reaction time can be about 4 hours. In another embodiment, the reaction time can be up to about 4 hours. In another embodiment, the reaction time can be from about 3 to about 5 hours. In another embodiment, the reaction time can be from about 2 to about 6 hours. In another embodiment, the reaction time can be from about 1 to about 8 hours. In another embodiment, the reaction time can be at least about 1 hour.
  • the addition of cellulase type enzymes to the PDMS can also increase the yield of 4-HBITC during this reaction phase,
  • a separation step can then be performed.
  • the separation step can be performed to separate the solvent from the reaction mixture such that the slurry is separated into a filter cake, an essential oil, and a residual solvent, wherein the essential oil and residual solvent can be an essential oil enriched solvent that is then separated into an essential oil and a residual solvent.
  • the slurry can be separated.
  • the slurry can be separated into a filter cake, an essential oil, and a residual solvent, wherein the essential oil and residual solvent can be an essential oil enriched solvent that is then separated into an essential oil and a residual solvent.
  • the solvent such as ethyl acetate if used, containing all of the 4-HBITC can be separated from the wet mustard filter cake and can then be further separated as described hereinafter to produce the essential oil.
  • This separation can be achieved by any separation technique as known in the art.
  • separation techniques can include centrifugation or filtration.
  • filtration, batch, or continuous centrifugation can be performed.
  • the separation can be performed immediately following the reaction described above. In another embodiment, the separation can be performed within about 1 hour following the reaction. In another embodiment, the separation can be performed with about 2 hours following the reaction.
  • filtration can be performed within an enclosed filtration system.
  • an enclosed filtration system can be used because ethyl acetate is volatile and generally should be contained so as to minimize evaporation into the atmosphere.
  • Filters that contain the filter cake within an enclosed container can also be used because of the volatile nature of ethyl acetate.
  • Some embodiments include vertical and horizontal leaf filters, nutsche filters, candle filters, and filters of similar design where filtration can be achieved within a closed or contained environment.
  • centrifugation can be performed.
  • the wetted mustard slurry prior to separation can have from about 30% to about 39% removable solid (suspended solids) material. In one embodiment, the wetted mustard slurry prior to separation can have from about 32% to about 35% removable solid (suspended solids) material. In one embodiment, the wetted mustard slurry prior to separation can have from about 33% to about 34% removable solid (suspended solids) material.
  • one embodiment of separation can include a decanter centrifuge. Decanter centrifuges can comprise a rotating cylindrical horizontal bowl that has a cylindrical section at one end and a radially sloping conical section at the other end with an optional lesser sloping section in between the two sections described above.
  • a scroll can be integrated in the conical section of the bowl and can be driven separately.
  • the wet mustard and solvent mixture (the wet mustard slurry) can enter the separating space through a centrally arranged feed tube entering through the cylindrical section of the bowl.
  • the wet mustard slurry can be spun against the inner bowl wall under the action of centrifugal force.
  • the scroll which can rotate at a different speed than the bowl shell, can transport the separated mustard solids to the bowl cone where the solids can discharge at the end of the bowl through a discharge port.
  • the ethyl acetate solvent stream can also be simultaneously separated and can exit the decanter at the cylindrical end.
  • the discharged wet mustard solids can drop directly onto a continuous belt dryer operating at a slight vacuum.
  • the temperature within the dryer can be kept at from about 70°C to about 75°C so that the ethyl acetate followed by the water evaporates from the wet mustard.
  • the slight vacuum can create a sufficient draft to force the ethyl acetate vapor and water vapor to move towards the vacuum source.
  • the output vapor containing the ethyl acetate vapor and water vapor can both be impinged on a cooling coil that condenses both the vapors to their liquid forms.
  • the condensed liquids can be pumped away to a separating tank where the ethyl acetate and water are separated. Ethyl acetate can be recycled to extract the next lot of mustard seed.
  • the wet mustard can be dried and desolventized completely in this vacuum dryer.
  • the wet mustard cake can be separated into a deheated and defatted mustard flour, as described herein, and a residual solvent.
  • the mustard flour can be used as described herein.
  • the ethyl acetate after centrifuging out the residual wet mustard cake, can be dried over a drying agent such as anhydrous sodium sulfate to remove the residual water, before evaporation.
  • a drying agent such as anhydrous sodium sulfate
  • the mustard solids can be separated from the solvent stream.
  • the solvent stream can then comprise an enriched solution of WMEO comprising 4-HBITC in solvent, such as ethyl acetate, and can then be stored in a tank prior to evaporation into an essential oil and a residual solvent.
  • further separation of the solvent, such as ethyl acetate, from the solvent stream can be performed to yield a residual solvent and an essential oil comprising WMEO comprising 4-HBITC.
  • evaporation can be performed under reduced pressure and temperature to avert the deleterious thermal effects on WMEO.
  • vacuum evaporation can be used.
  • the evaporation can remove about 99% or more of the ethyl acetate in a single pass via the evaporator and thus minimization of the thermal degradation effects on WMEO and hence 4-HBITC can occur.
  • a range of evaporators can be used for this purpose. Rising film evaporators, falling film evaporators, centrifugal evaporators are evaporators that can be used.
  • this evaporation can result in an essential oil.
  • white mustard essential oil comprising from about 5% to about 35% 4-hydroxybenzyl isothiocyanate (4-HBITC) is provided.
  • WMEO comprising from about 10% to about 30% 4-HBITC is produced.
  • WMEO comprising from about 15% to about 27% 4-HBITC is produced.
  • WMEO comprising from about 22% to about 28% 4-HBITC is produced.
  • the WMEO can be further purified by first mixing it with hexane, heptane, or methyl pentane at a ratio of one part WMEO to about 1.2 to about 1.3 parts hexane, heptane, or methyl pentane. Such mixing can remove some of the triglyceride materials in the WMEO and can result in separating the lower oil layer containing most of the 4-HBITC from the hexane.
  • the hexane layers can then be pooled and repeatedly extracted with absolute methanol to remove the 4-HBITC in the hexane layer.
  • the methanol layers and the original lower oil layer can then combined and evaporated under reduced pressure to yield a highly enriched WMEO with from about 30% to about 80% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 35% to about 75% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 45% to about 70% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 49% to about 65% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 45% to about 55% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 35% to about 80% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 40% to about 80% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 45% to about 80% 4-HBITC, by weight.
  • the highly enriched WMEO can comprise from about 50% to about 80% 4-HBITC, by weight. This highly purified WMEO can then be suitable for addition to food and beverage products requiring a higher flavor quality.
  • the WMEO can then be mixed at a suitable ratio with a water soluble hygroscopic substance such as maltodextrin. Because the WMEO is now enriched in 4-HBITC, the ratio of WMEO to maltodextrin is less than 1:9. In one embodiment, the range can be from about 1:6 to about 1:9.
  • the essential oil itself which contains one or more moisture sensitive isothiocyanate compounds, preferably 4-hydroxybenzyl isothiocyanate, may then be utilized in the compositions and methods as described in, for example, US 6,361,812B1; US 6,558,723B2; US 7,105, 190B2 and US 7,658,961.
  • additional processing can be performed to the WMEO and/or the 4-HBITC.
  • freezing of the WMEO can be performed. Such freezing can preserve the essential oil. Freezing can be performed down to about -25°C. After freezing, the WMEO can be plated into maltodextrin, or any other hygroscopic carrier, and used as described in, for example, US 6,361,812B1; US 6,558,723B2; US 7,105,190B2 and US 7,658,961.
  • the concentration of 4-hydroxybenzyl isothiocyanate in white mustard essential oil is dependent on the fat content of the starting partially defatted white mustard press cake. This property holds because the solvent dissolves all fat soluble materials from the resulting moist mustard flour, and the composition of the white mustard essential oil is the sum total of the 4-hydroxybenzyl isothiocyanate and the residual fat soluble material in the starting partially defatted white mustard press cake.
  • the concentration of 4-HBITC in WMEO is about 5-6%; when partially defatted white mustard powder with a fat content of about 17.5% is used as the starting material the concentration of 4-HBITC in WMEO is about 10.1%; when the fat content is about 14% in the starting partially defatted white mustard the concentration of 4-HBITC in WMEO is about 15.5-15.9%; when the fat content of the starting defatted white mustard is about 8.2%, the concentration of 4-HBITC is about 24% in the resulting WMEO; finally when the fat content is about 6-7% in the starting partially defatted white mustard, the 4-HBITC concentration in WMEO is about 26%.
  • the fat content in the starting partially defatted white mustard is approximately linearly correlated to the concentration of 4-HBITC in WMEO. If the starting white mustard press cake is fully defatted then the concentration of 4-HBITC in WMEO can reach as high as about 90%.
  • the inventors have surprisingly found that the range of antimicrobial effectiveness of WMEO is dependent on the concentration of 4-hydroxybenzyl isothiocyanate in it. For example, WMEO, with a 4-hyroxybenxzyl isothiocyanate concentration of about 5-6 %, can be effective against gram negative organisms only and has been found to have substantially no effect on the growth of gram positive organisms.
  • 4-hydroxybenzyl isothiocyanate concentration in WMEO is at about 15%, a noticeable but limited inhibitory effect against gram positive organisms in addition to the sustained inhibitory effect on gram negative organisms has been observed.
  • the 4-hydroxybenzyl isothiocyanate concentration in WMEO is at about 24%, a very noticeable inhibitory effect against both gram negative and gram positive organisms has been observed.
  • the inventors think that the presence of fixed mustard oil along with 4-HBITC in the WMEO exerts a partitioning effect on the 4-HBITC, which can slow or prevent its release into the aqueous media, which is required for action against microorganisms.
  • Gram negative microorganisms have a cell wall composed of lipo- poly saccharides while gram positive microorganisms have a cell wall composed mostly of peptidoglycan type molecules.
  • hydrophobic compounds such as 4-HBITC
  • the WMEO can be further purified by first mixing it with hexane or methyl pentane at a ratio of 1 part WMEO to about 1.2 to about 1.3 parts hexane or methyl pentane, to remove some of the triglyceride materials in the WMEO, and separating the lower oil layer containing most of the 4-HBITC from the hexane. This extraction can be repeated one to two more times to ensure that the WMEO is further enriched.
  • the hexane layers can then pooled and repeatedly extracted with absolute methanol to remove the 4-HBITC in the hexane layer.
  • the methanol layers and the original lower oil layer is then combined and evaporated under reduced pressure to yield a highly enriched WMEO with greater than 50% 4-HBITC.
  • This highly purified WMEO is suitable for addition to food and beverage products requiring a higher flavor quality.
  • the WMEO can then mixed at a suitable ratio with a water soluble hygroscopic substance such as maltodextrin. Because the WMEO is now enriched in 4-HBITC the ratio of WMEO to maltodextrin can be less than about 1:9.
  • the resulting dried, deheated, and defatted mustard flour can have a protein content of about 42%, or from about 35% to about 45%, or from about 35% to about 45%, or from about 38% to about 43%.
  • the flour can have a fat content of about 2.5%, or from about 1% to about 5%, and from about 2% to about 4%.
  • the mustard seed mucilage having emulsification properties is not altered as a result of processing to remove the white mustard essential oil and the resulting deheated and defatted mustard flour can be used for the original intended purpose as an emulsifier for meat products, with the additional benefit of being low in fat.
  • the deheated and defatted mustard flour as disclosed herein will not generate any of the "heat” when contacted with other foods sources.
  • the high protein content of the deheated & defatted mustard flour can lend itself to other uses as a protein source in protein enriched products.
  • Prior approaches to making use of mustard protein have to do with extracting the protein component out of the mustard seed with the use of various solutions, thus decoupling the emulsification property from the protein component.
  • the approach described herein allows the flexibility for a food processor to make use of the whole deheated & defatted mustard or of the protein separately without the risk of generating objectionable "heat” by accidental activation of the myrosinase system.
  • one embodiment of the present invention relates to a deheated and defatted mustard flour.
  • defatted and deheated mustard flour After drying the wet mustard press cake resulting from the separation step above, defatted and deheated mustard flour is formed in good yield.
  • This deheated and defatted mustard flour has no sinalbin present as all of it has been hydrolyzed to 4-hydroxybenzyl isothiocyanate and the myrosinase enzyme is largely denatured as a result of the drying step.
  • one embodiment includes a deheated and defatted mustard flour.
  • the deheated and defatted mustard flour can be substantially free of sinalbin, and/or substantially all of the myrosinase enzyme can be denatured.
  • the emulsification properties associated with mustard flour to do with its mucilage is well preserved in the deheated & defatted mustard flour.
  • the resulting deheated and defatted mustard flour can have a protein content of about 42%, or from about 35% to about 45%, or from about 35% to about 45%, or from about 38% to about 43%.
  • the flour can have a fat content of about 2.5%, or from about 1% to about 5%, and from about 2% to about 4%. Because the deheated and defatted mustard flour is substantially devoid of sinalbin and/or the myrosinase enzyme has been denatured, the heat sensation associated with wetted mustard flour will not arise.
  • the mustard flour can be used in many situations requiring an emulsifier or a high protein component.
  • the flour can be used as a replacer of currently used mustard flour.
  • Specific examples include in meat emulsions, such as sausages; as a high protein source similar to soy and canola meal in high protein bar type products; and in pet foods as a high protein component.
  • this deheated and defatted mustard flour can be quite economical considering that several high value components are derived from it. IV. Test Method For Measurement of 4-Hydroxybenzyl Isothiocyanate and Identification of Moisture sensitive Isothiocyanate Compounds
  • Supercritical fluid chromatography can be used to determine the amount of
  • 4-hydroxybenzyl isothiocyanate in a preservative composition First an accurately weighed amount of the preservative composition is either dissolved in a compatible solvent such as ethyl acetate or ethyl acetate and ethanol mixtures or solid preservative compositions are extracted repeatedly with ethyl acetate.
  • a compatible solvent such as ethyl acetate or ethyl acetate and ethanol mixtures or solid preservative compositions are extracted repeatedly with ethyl acetate.
  • the compound is further purified by flash column chromatography as described by Still, W. C, Kahn, M. and Mitra A.. J. in " Rapid chromatographic method for separation with moderate resolution," Organic Chemistry, 14, pp. 2923-2925. 1978, with modifications.
  • the reaction product dissolved in mobile phase (4mL) is placed on top of the column. Elution is performed by adjusting the argon overpressure in a way so that the solvent head drops about 2 inch/min. Aliquots of lOmL are collected.
  • the target compound usually elutes in fractions 6-10.
  • the fractions are combined and after removing the solvent by rotary evaporation at 45°C in vacuum, a yellow oil is obtained (typical yield: 66%).
  • the structure of 4-hydroxybenzyl isothiocyanate is confirmed by H ⁇ NMR in CDC1 3 , C 13 -NMR in CDCl 3 and GC-MS operating in electron impact mode.
  • Moisture sensitive isothiocyanate compounds are identified by suspending the isothiocyanate containing material in an aqueous phosphate buffer (pH of about 3.6) at room temperature. The resulting suspension is shaken well, and a time zero sample is withdrawn into a separatory funnel and extracted with ethyl acetate. This extraction is repeated with two further volumes of ethyl acetate. The separated ethyl acetate layers are pooled and dried over anhydrous sodium sulfate and kept frozen before analysis of the time zero isothiocyanate concentration by supercritical fluid chromatography.
  • aqueous phosphate buffer pH of about 3.6
  • the isothiocyanate suspension is stored at a temperature between about 20°C to about 23°C for a period of about 24 hours.
  • the extraction procedure is repeated after 24 hours and the level of residual isothiocyanate compound is measured by supercritical fluid chromatography.
  • Moisture sensitive isothiocyanate compounds are characterized by at least about a 20% reduction in concentration of the isothiocyanate after about 24 hours, relative to the time zero or starting concentration.
  • a white mustard essential oil is generated by adding water to ground white mustard seeds and then extracting the essential oil with supercritical carbon dioxide according to the known processes described in the art. Immediately after extraction, residual moisture is removed from the essential oil by centrifugation and by drying under vacuum. The resulting white mustard essential oil contains about 25% by weight 4-hydroxybenzyl isothiocyanate.
  • the essential oil is suspended in an aqueous phosphate buffer (pH of about 3.6) at room temperature (about
  • White mustard essential oil was prepared by first cold pressing 800 kg of white mustard seed using a Rosedowns Mini 200 screw press unit. The resulting press cake exited the press at about 75-78°C and was immediately cooled to room temperature by hammer milling. The oil content of the press cake was 17%. About 340 kg from the hammer milled white mustard press cake was introduced into a ribbon blender and 78 g ascorbic acid added to it while blending to ensure uniform mixing. About 102 kg of tap water at room temperature was added in small proportions to ensure uniform wetting of the press cake. After mixing for about 10-15 minutes, the moistened and myrosinase enzyme activated press cake was transferred to a stirred solvent tank containing 626 kg ethyl acetate.
  • the moistened, activated mustard press cake and ethyl acetate slurry was stirred within the closed solvent tank for about 4 hours at room temperature, to ensure generation and transfer of the 4-hydroxy benzyl isothiocyanate from the moist white mustard press cake into the ethyl acetate.
  • the slurry of moist white mustard press cake in ethyl acetate was pumped into a decanter centrifuge to separate the ethyl acetate from the moist white mustard press cake.
  • the ethyl acetate layer was collected in a static solvent tank while the wet white mustard press cake with some residual ethyl acetate was directly transferred into a vacuum assisted belt dryer.
  • the vacuum conditions allowed the moist ethyl acetate to leave the mustard press cake and get condensed in an external condenser.
  • the ethyl acetate top layer was separated out and stored for re-use while the water layer was discharged.
  • the mustard press cake continued to travel on a continuous belt towards the bottom of the dryer where the dried, solvent free mustard press cake exited via a screw conveyor.
  • This deheated and defatted mustard flour was analyzed for its gross composition and its amino acid composition.
  • the ethyl acetate layer containing the WMEO was pumped into a rising film evaporator kept under about 56 cm Hg vacuum to remove the ethyl acetate from the white mustard essential oil.
  • the evaporation step resulted in about 65.5 kg of WMEO containing 8.4% 4-hydroxybenzyl isothiocyanate.
  • White mustard essential oil was prepared by first cold pressing 5 kg of white mustard seed using a model KK8 single screw press (KERNKRAFT, Moosbauer and Rieglsperger GbR. Germany) to yield a white mustard press cake with 6.8% fixed oil and a temperature of about 60°C.
  • the press cake was hammer milled to provide a uniform powder and 151.5 g of the powder weighed out into a weighing pan.
  • a closed mixing vessel equipped with a top drive mixer 45.5 g of tap water was mixed with 305 mL of ethyl acetate and 0.036 g of ascorbic acid added while mixing.
  • the partially defatted mustard powder in the weighing pan was added to the mixing vessel and mixed at high speed to ensure uniform dispersion. When a uniform slurry had formed the speed was slowed down and the mixing continued for a further 4 hours. The slurry was removed from the mixing vessel at that time and centrifuged to separate the ethyl acetate from the moistened, partially defatted mustard press cake. The ethyl acetate was decanted from the centrifuge tubes and evaporated under reduced pressure to yield about 9.5 g white mustard essential oil containing 27%
  • a powdered preservative composition is prepared by triturating the white mustard essential oil of Example 2 with maltodextrin according to the following formulation.
  • the blend of materials is intimately mixed or triturated using a mortar and pestle.
  • the level of 4-hydroxybenzyl isothiocyanate in the resulting preservative composition is about 0.84% by weight.
  • the level of 4-hydroxybenzyl isothiocyanate in the powdered preservative composition remains stable during storage of the preservative composition at room temperature (about 21.1 °C).
  • a powdered preservative composition is prepared by triturating the white mustard essential oil of Example 3 with maltodextrin according to the following formulation.
  • the blend of materials is intimately mixed or triturated using a mortar and pestle.
  • the level of 4-hydroxybenzyl isothiocyanate in the resulting preservative composition is about 2.6% by weight.
  • the level of 4-hydroxybenzyl isothiocyanate in the powdered preservative composition remains stable during storage of the preservative composition at room temperature (about 21.1 °C).
  • Peptone broth (0.5% by weight, pH 7.1) was inoculated with 20-24 hour old cultures of Staphylococcus aureus (ATCC 6538), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (ATCC 7644) grown on trypticase soy agar slants (incubated at 35°C) and Clostridium perfringens (ATCC 3624) cultures grown on Shahidi Ferguson perfringens (SFP) agar slants incubated under anaerobic conditions at 35°C. Starting counts were between lxlO 5 and lxlO 7 cfu mL 1 .
  • Example 4 The powdered preservative composition of Example 4 was introduced into the inoculated media to reach a starting 4-HBITC level of 350 mg L "1 and shaken well. Samples were stored at 6.5°C and plated on microbial content test (MCT) agar at 1 day after neutralization with Letheen as per USP 26 (United States Pharmacopoeia, Rockville, MD, USA). The mean log reductions in microbial counts were 2.23 for 5. aureus, 1.61 for 5. enteritidis, 0.80 for L. monocytogenes and 2.17 for C. perfringens.
  • MCT microbial content test
  • Peptone broth (0.5% by weight, pH 7.1) was inoculated with 20-24 hour old trypticase soy agar slants (incubated at 35°C) of Staphylococcus aureus (ATCC 6538), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (ATCC 7644).
  • Clostridium perfringens (ATCC 3624) cultures were grown on Shahidi Ferguson perfringens (SFP) agar slants and incubated under anaerobic conditions at 35°C. Starting counts were between lxlO 5 ⁇ lxlO 7 cfu mL "1 .
  • Example 5 The powdered preservative composition of Example 5 was introduced into the inoculated media to reach a starting 4-HBITC level of 360 mg L "1 and shaken well. Samples were stored at 6.5°C and plated on microbial content test (MCT) agar at 1 day after neutralization with Letheen as per USP 26 (United States Pharmacopoeia, Rockville, MD, USA). The mean log reductions in microbial counts were 3.95 for S. aureus, 4.93 for S. enteritidis, 4.17 for L. monocytogenes and 5.40 for C. perfringens.
  • MCT microbial content test

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Abstract

La présente invention concerne un procédé de production d'une huile essentielle. Cette huile essentielle peut être l'huile essentielle de moutarde blanche. L'huile essentielle de moutarde blanche peut comporter un composé isothiocyanate sensible à l'humidité. Le composé isothiocyanate sensible à l'humidité peut être le 4-HBITC. L'huile essentielle peut être produite à partir de graine de moutarde comprenant un précurseur de la sinalbine, et une enzyme, la myrosinase. La moutarde peut être réduite en poudre. En utilisant un solvant aqueux et un promoteur permettant de former une suspension épaisse, il est possible de réaliser l'activation de l'enzyme myrosinase, laquelle enzyme myrosinase catalyse la production d'une huile essentielle comprenant un isothiocyanate à partir du précurseur de la sinalbine.
EP11712387A 2010-03-31 2011-03-22 Procédé d'extraction d'isothiocyanates Withdrawn EP2552467A1 (fr)

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CN102811727A (zh) 2012-12-05
US20110245526A1 (en) 2011-10-06
AU2011233581B2 (en) 2014-06-05
CA2793529A1 (fr) 2011-10-06
AU2011233581A1 (en) 2012-10-25
MX2012011357A (es) 2012-11-12
BR112012024699A2 (pt) 2016-06-07
WO2011123280A1 (fr) 2011-10-06
US8697150B2 (en) 2014-04-15

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